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Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFkappaB.

ABSTRACT: We describe a highly efficient use of lentiviral validation-based insertional mutagenesis (VBIM) to generate large populations of mammalian cells in which a strong promoter is inserted into many different genomic loci, causing greatly increased expression of downstream sequences. Many different selections or screens can follow, to isolate dominant mutant clones with a desired phenotypic change. The inserted promoter can be excised or silenced at will, to prove that the insertion caused the mutation. Cloning DNA flanking the insertion site identifies the locus precisely. VBIM virus particles are pseudotyped with VSV G protein, allowing efficient infection of most mammalian cell types, including non-dividing cells, and features are included that give high yields of stable virus stocks. In several different selections, useful mutants have been obtained at frequencies of approximately 10(-6) or higher. We used the VBIM technique to isolate mutant human cells in which the F-box leucine-rich protein 11 (FBXL11), a histone H3K36 demethylase, is shown to be a negative regulator of NFkappaB. High levels of FBXL11 block the ability of NFkappaB to bind to DNA or activate gene expression, and siRNA-mediated reduction of FBXL11 expression has the opposite effects. The H212A mutation of FBXL11 abolishes both its histone H3K36 demethylase activity and its ability to inhibit NFkappaB. Thus, we have used a powerful tool for mutagenesis of mammalian cells to reveal an aspect of the complex regulation of NFkappaB-dependent signaling.

SUBMITTER: Lu T

PROVIDER: S-EPMC2736141 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundDysfunctional CFTR in the airways is associated with elevated levels of NFkappaB mediated IL-8 signaling leading to neutrophil chemotaxis and chronic lung inflammation in cystic fibrosis. The mechanism(s) by which CFTR mediates inflammatory signaling is under debate.Methodology/principal findingsWe tested the hypothesis that wt-CFTR down-regulates NFkappaB mediated IL-8 secretion. We transiently co-expressed wt-CFTR and IL-8 or NFkappaB promoters driving luciferase expression in HEK293 cells. Wt-CFTR expression in HEK293 cells suppresses both basal and IL1beta induced IL-8, and NFkappaB promoter activities as compared to the control cells transfected with empty vector (p<0.05). We also confirmed these results using CFBE41o- cells and observed that cells stably transduced with wt-CFTR secrete significantly lower amounts of IL-8 chemokine as compared to non-transfected control cells. To test the hypothesis that CFTR must be localized to cell surface lipid rafts in polarized airway epithelial cells in order to mediate the inflammatory response, we treated CFBE41o- cells that had been stably transduced with wt-CFTR with methyl-beta-cyclodextrin (CD). At baseline, CD significantly (p<0.05) induced IL-8 and NFkappaB reporter activities as compared to control cells suggesting a negative regulation of NFkappaB mediated IL-8 signaling by CFTR in cholesterol-rich lipid rafts. Untreated cells exposed to the CFTR channel blocker CFTR-172 inhibitor developed a similar increase in IL-8 and NFkappaB reporter activities suggesting that not only must CFTR be present on the cell surface but it must be functional. We verified these results in vivo by comparing survival, body weight and pro-inflammatory cytokine response to P. aeruginosa LPS in CFTR knock out (CFKO) mice as compared to wild type controls. There was a significant (p<0.05) decrease in survival and body weight, an elevation in IL-1beta in whole lung extract (p<0.01), as well as a significant increase in phosphorylated IkappaB, an inducer of NFkappaB mediated signaling in the CFKO mice.Conclusions/significanceOur data suggest that CFTR is a negative regulator of NFkappaB mediated innate immune response and its localization to lipid rafts is involved in control of inflammation.

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Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFkappaB.

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